1

The Impact of Caffeine on Dehydration in Naked Eggs

Jashan T.

Toor.jashan.wjm@gmail.com

Abstract:

Dehydration can cause undesired side effects, especially when it is paired with an intensive exercise such as weightlifting. While weightlifters often drink water along with their workout, pre-workout supplements with large amounts of caffeine have become a staple in weightlifting. This study aimed to investigate the impact of caffeine on dehydration in naked eggs as human trials were not feasible at the time of this study. Dehydration was attempted to be measured as the percent change in weight experienced by the naked eggs. A one-way ANOVA test was conducted, which yielded a p-value of 0.5232. This is far greater than this study’s significance level of 0.05, elucidating that there was no statistically significant difference in dehydration between the treatment groups. Errors in the experiment set up did not allow for the impact of caffeine on dehydration to be investigated as intended. This study will also provide suggestions on how this error can be avoided in future research.

Introduction:

Pre-workout supplements (referred to as PWS hereafter) are a common addition to a

weightlifter’s pre-workout routine that promote elevated levels of energy as a result of their high

caffeine content. The global PWS market is valued at 12.6 billion USD and is expected to grow

at an annual rate of 8.3% from 2020 to 2027 (Grandview research). In Canada it was found that

It was found that 86% of PWS contain caffeine (Jagim et al. 254) with the average dose being

150-300mg per serving (Hollowc2) while some PWS can reach up to 600mg per serving

(“HOOLIGAN Pre Workout...”). As a result of having large amounts of caffeine in these

supplements, their safety is often debated. Dehydration is an accompanying risk that users of

2

these supplements face and is listed as a side effect for many PWS (Weik). The impact that

caffeine has on dehydration is a topic that has been well researched and has even been correlated

to exercise. A previous paper looked at the effect of espresso with 171 ± 8.9 mg of caffeine and

found that dehydration occurred to 1.2% of the individual's body weight (Zavvos et al. 288).

Another paper done by Zhang et al. specifically looked at caffeine ingestion prior to exercise and

they found that at a median caffeine dosage of 300mg the diuretic effect caused by caffeine was

negated during exercise so excess fluid loss was not exhibited in the 379 participants while

exercising (570-573). However, both of these studies leave gaps on what happens if caffeine is

taken in excess of the 400mg daily allowance (Food and Drug Administration), which can be the

case with these pre workout supplements as they can be taken in 600mg doses. Since human

trials were not feasible, this study will attempt to mimic the impact that caffeine has on

dehydration through observing eggs with a dissolved shell (hereafter referred to as naked eggs).

The weight loss that occurs in the naked eggs will be a result of osmosis, where water moves

from the egg to the surrounding hypertonic environment. This is intended to mimic caffeine

entering the bloodstream and drawing water out of muscles because as Hill et al. state, muscles

can retain water as a result of increased levels of cortisol due to medium to high intensity

exercise (587), therefore increasing the water content in the muscles and helping establish an

osmotic gradient.

This study will attempt to answer if caffeine impacts dehydration in naked eggs. If

caffeine causes dehydration, then there should be a greater weight loss in naked eggs that have

been immersed in the solution containing 600mg of caffeine when compared to those that have

3

been immersed in solutions containing lower levels of caffeine. If the results found are

significant then the null hypothesis which states there is no difference in egg weight loss through

dehydration when submerged in solutions with varying caffeine concentrations will be rejected

and the alternate hypothesis that there is a difference in egg weight loss via dehydration when

submerged in solutions with varying caffeine concentrations will be supported.

Methods:

12 clear plastic cups were filled with 300mL of vinegar each and one egg was submerged

in each cup. The eggs were left to sit in the vinegar solution for 24 hours so that the eggshell

would dissolve completely. The formation of bubbles on the eggshells indicated that the reaction

had started. As seen in equation 1, these bubbles are carbon dioxide bubbles that formed as a

result of the reaction between the calcium carbonate eggshells and acetic acid in the vinegar.

2CH3COOH (l) + CaCO3 (s) g H2O (l) + CO2 (g) + Ca(CH3COO)2 (s)

Equation 1: This equation shows the balanced reaction between the calcium carbonate in the

eggshells and the surrounding vinegar environment to create carbon dioxide and calcium acetate

(“Naked Eggs”).

After the 24 hours the eggs were removed from the cup and dried using paper towels. A

clear plastic cup was then placed on the digital scale and the scale was zeroed with the cup on it

to allow the mass of the eggs to be measured without having to subtract the mass of the cup after

4

each measurement. Each egg was placed into the cup one at a time and the mass was recorded as

the initial weight. The plastic cups were emptied, and the eggs were placed into a temporary

storage container (Figure 1). As shown in Figure 1 below, the eggs had left over eggshell

residue, which according to equation 1 was calcium acetate, this residue was wiped off the egg

using paper towels.

Figure 1: The eggs after their eggshells were dissolved in a vinegar solution placed in their

temporary storage container. The white dots on the eggs are left over pieces of calcium acetate,

this was wiped off prior to placing them in their respective treatment.

12 empty plastic cups were labelled with the sample number and the solution they were

going to contain. Cups 1-3, were labelled as 500mL of vinegar, 4-6 were labelled as 200mg of

caffeine, 7-9 were labelled as 400mg of caffeine and 10-12 were labelled as 600mg of caffeine. A

5

tinfoil piece was folded on three edges to make a boat to weigh the caffeine. 50 Allmax Caffeine

pills containing 200mg of caffeine each were crushed in a mortar and pestle to turn them into a

fine powder. The tinfoil boat was placed on the digital scale and the scale was zeroed to account

for the mass of the tinfoil boat. 600mg of the powdered caffeine was weighed out onto the tinfoil

boat using a teaspoon and the caffeine was then put into a clear plastic cup that was labelled with

the sample number and amount of caffeine. This was done for the 200mg and 400mg caffeine

samples as well and was repeated three times for each caffeine quantity. In total, there were 3

cups containing 600mg of caffeine, 3 cups containing 400mg of caffeine, and 3 cups containing

200mg of caffeine. Afterwards 500mL of water was added to each cup with caffeine, cups 4-12.

The final concentrations of the caffeine containing solutions were 1.2mg/mL for the cups

containing 600mg of caffeine, 0.8mg/mL for the cups containing 400mg of caffeine and 0.4mg/

mL for the cups containing 200mg of caffeine. Afterwards, 500mL of vinegar was added to cups

1-3. The vinegar only treatment acted as the control for the study as the vinegar was intended to

be isosmotic with the eggs therefore there should not have been weight loss or weight gain

observed in the naked eggs in these cups. By having the vinegar only solution as the control, the

amount of caffeine was intended to be the only variable that would impact the weight of the

naked eggs so any statistically significant differences in the weight of the eggs would be a result

of the caffeine content in each treatment. The cups with caffeine and water were stirred with a

teaspoon to help dissolve the caffeine powder. After the caffeine had dissolved into the solutions

(Figure 2), the eggs were left in their respective solutions for 24 hours and the cups were placed

in a storage container so they would not be disturbed.

6

Figure 2: This figure shows naked eggs numbered 8-10 and the amount of caffeine in that

treatment after being placed in their storage container for 24 hours. The solutions have been

created using caffeine and water, sample 8 and 9 have a caffeine concentration of 0.8mg/mL

while sample 10 has a caffeine concentration of 1.2mg/mL. The naked eggs were visibly larger

than they were in Figure 1 prior to being subjected to their respective treatments.

After 24 hours, the cups were removed from the storage container. One clear and empty

plastic cup was placed on the digital scale and the scale was zeroed. The naked eggs were

removed from the solution one at a time and their exterior was dried with paper towels. The dried

eggs were then placed in the plastic cup on the scale and the weight was recorded as the final

weight. This was done for all 12 eggs. After the weight of all the eggs was recorded, the

7

solutions, plastic cups, and eggs were disposed of. After compiling the data, the percent change

in weight was calculated and this data was used for the statistical tests.

Results:

Each treatment group had a sample size of 3 (n=3), for a total of 12 samples. Each

sample gained weight and the mean percent change in egg mass was 12.2% for the control group,

15.1% for the 200mg group, 14.9% for the 400mg group and 15.8% for the 600mg group. The

95% confidence interval for the control group was 9,0% to 15.4%, for the 200mg group was

10.3% to 19.9%, for the 400mg was 13.2% to 16.6% and for the 600mg group was 12.7% to

18.9%. Sample 3, which had a percent change in weight of 8.9% did not fit into the confidence

interval for the control group and sample 12, which had a percent change in weight of 12.6% did

not fit into the confidence interval for the 600mg group as seen in figure 3.

8

Figure 3: Percent change for each naked egg sample and mean percent change for each treatment

group. The sample size is n=3 for each treatment group. The mean percent change in weight for

the control group is observed to be lower than the caffeinated samples. The largest confidence

intervals are seen in the 200mg group and the smallest confidence intervals are seen in the

400mg group indicating that the 400mg group measurements were more precise than those of the

other groups. This data was used to conduct a one-way ANOVA test which returned a p-value of

0.5232.

The alpha/significance level for this study was set at 0.05 because any results obtained

with a p-value below this had a greater than 95% probability that the null hypothesis is incorrect

and that the results obtained are statistically significant. This was also chosen as the significance

level because previous literature done by Zavvos as well as Zhang used 0.05 as the significance

9

level (Zavvos et al. 288; Zhang et al. 570), so by using 0.05 in this study, it would be easier to

directly compare results. As a result, the p-values obtained from the statistical tests must be less

than 0.05 for the null hypotheses of those tests to be rejected and the alternate hypotheses

supported. A Shapiro-Wilk test was conducted to see if the samples came from a normal

distribution and it provided four p-values, one for each treatment. The p-value for the vinegar

only solution was 0.1666, for the 200mg caffeine solution was 0.5439, for the 400mg solution

was 0.3212 and for the 600mg solution was 0.3106. A one way ANOVA was conducted on the

percent change data to determine if the data means of the groups were significantly different

from each other and a p-value of 0.5232 was observed.

Discussion:

In this study, the impact of caffeine on dehydration was tested in naked eggs It was found

that as a result of the experimental setup, weight loss in naked eggs was unable to be recorded as

the eggs gained weight and yielded data which were not significant according to the statistical

tests conducted.

Ten out of the twelve percent change values were within their treatment group’s 95%

confidence intervals but sample 3, part of the control group, and sample 12, part of the 600mg

group, were not so there is a greater than 95% probability that these values are not indicative of

the true mean of their groups. The Shapiro-Wilk test p-values were greater than the significance

level of 0.05 used in this paper, therefore the null hypothesis of the Shapiro-Wilk test, that the

10

data come from a normal distribution, was unable to be rejected and the alternate hypothesis, that

says the data does not come from a normal distribution, was unable to be supported by these

results. This allowed for a one-way ANOVA test to be conducted on the normally distributed data

for percent change in egg mass as the assumptions of the one-way ANOVA were met. The one-

way ANOVA yielded a p-value of 0.5232 which is greater than the significance level of 0.05

therefore the null hypothesis that there is no difference in egg weight loss through dehydration

when submerged in solutions with varying caffeine concentrations, was not rejected. This also

meant that the alternate hypothesis that there is a difference in egg weight loss via dehydration

when submerged in solutions with varying caffeine concentrations was unable to be supported.

As a result this disproves the original hypothesis which was if caffeine has an impact on

dehydration, then there should be a greater weight loss in the naked eggs submerged in the

600mg caffeine solution than the other treatment groups. This demonstrates that there was not a

significant difference seen between the means of the treatment groups so it could not be

concluded that one group has a greater impact on dehydration than the other groups.

This experiment was intended to demonstrate weight loss in the naked eggs however, no

weight loss was observed in the naked eggs. Instead all of the naked eggs ended up gaining

weight. This could be a result of incorrectly assuming that the naked eggs would be isotonic or

hypotonic to their environment. Since the pure white vinegar was 93-96% water (Hill) and the

eggs are 74% water (Johnson and Ridlen), it was assumed that this would be enough to change

the osmolarity of the eggs to the point that they would be hypotonic or isotonic compared to their

environment. However, it was evident that this was not the case and that this was an incorrect

11

assumption to make. A previous experiment found that naked eggs increased in size when they

were placed in their initial vinegar solution as a result of water moving from the vinegar into the

egg (Science World). This movement of water would also result in weight gain which is similar

to what was observed in the results for this study. Another paper by Cocanour and Bruce also

found that naked eggs gained weight when submerged in water, they also found that eggs placed

in a glucose solution lost weight as water moved out of the egg and into the surrounding

environment (127-130). Previous literature does not discuss the impact of caffeine on naked eggs

and whether that could have influenced the obtained results. However, since weight loss was

observed when the egg was placed in a glucose solution, it is possible that submerging naked

eggs in a solution containing caffeine would also result in water moving out of the eggs and

therefore causing dehydration to occur in the eggs.

To truly test if caffeine impacts dehydration through weight loss in naked eggs, future

research must have a 48 hour period of submerging the naked eggs in distilled water prior to the

caffeine treatment to ensure that the eggs will be hypotonic to their environment. This may have

prevented the unexpected weight gain that was observed in this experiment and could have led to

more significant results on the impact of caffeine on dehydration. This experiment could be

repeated by just making this alteration but the sample size could also be increased to raise the

statistical significance of the results obtained by the scientists doing this experiment in the future.

12

Conclusion:

This experiment attempted to demonstrate the impact that caffeine has on dehydration in

naked eggs but as a result of errors in the methods this experiment was unsuccessful in

demonstrating that a link between caffeine and dehydration measured through the percent change

in weight exists in naked eggs. The p-values obtained from the one-way ANOVA were greater

than the significance level of 0.05 therefore, the mean percent change in weight of the treatment

groups were not significantly different from one another.

Acknowledgements:

It is acknowledged that the BIOL 342 course is held at UBC on the traditional, ancestral,

and unceded territory of the xʷməθkʷəy̓əm (Musqueam) First Nation. Further acknowledgements

go out to Dr. Celeste Leander and the teaching team for their help in critiquing the research

proposal for this experiment and continued support in providing advice and expertise.

13

Works Cited

Cocanour, Barbara, and Alease S. Bruce. “Osmosis and The Marvelous Membrane.” Journal of

College Science Teaching, vol. 15, no. 2, 1985, pp. 127–130. JSTOR, www.jstor.org/

stable/42988703. Accessed 16 Apr. 2021.

Food and Drug Administration. “Spilling the Beans: How Much Caffeine Is Too Much?” U.S.

Food and Drug Administration, 12 Dec. 2018, www.fda.gov/consumers/consumer-

updates/spilling-beans-how-much-caffeine-too-much. Accessed 18 Apr. 20201.

Hill, Ansley. “White Vinegar: Ingredients, Uses and Benefits.” Healthline, 31 July 2018,

www.healthline.com/nutrition/white-vinegar. Accessed 18 Apr. 2021.

Hill, E. E., et al. “Exercise and Circulating Cortisol Levels: The Intensity Threshold Effect.”

Journal of Endocrinological Investigation, vol. 31, no. 7, 2008, p. 587. Crossref,

doi:10.1007/bf03345606. Accessed 14 Apr. 2021.

Hollowc2. “Does Taking a Pre-Workout Actually Work?” Health Essentials from Cleveland

Clinic, Cleveland Clinic, 15 Aug. 2019, health.clevelandclinic.org/does-taking-a-pre-

workout-actually-work/#:

%7E:text=Most%20brands%20of%20pre%2Dworkout,through%20a%20jolt%20of%20c

affeine. Accessed 17 Apr. 2021

14

Jagim, Andrew, et al. “Common Ingredient Profiles of Multi-Ingredient Pre-Workout

Supplements.” Nutrients, vol. 11, no. 2, 2019, p. 254. Crossref, doi:10.3390/nu11020254.

Accessed 18 Apr. 2021.

Johnson, H., and S. Ridlen. “Structure of the Egg - Incubation and Embryology - University of

Illinois Extension.” University of Illinois Incubation and Embryology,

web.extension.illinois.edu/eggs/res16-egg.html. Accessed 18 Apr. 2021.

Lift Vault. “Hooligan Pre Workout Review.” Lift Vault, Lift Vault, 18 Apr. 2021, liftvault.com/

nutrition/hooligan-pre-workout-review. Accessed 18 Apr. 2021.

“Naked Eggs.” Imagination Station, www.imaginationstationtoledo.org/education-resources/diy-

activities/naked-eggs/#:

%7E:text=If%20you%20soak%20this%20egg,as%20bubbles%20on%20the%20shell.

Accessed 13 Apr. 2021.

Science World. “Naked Eggs: Osmosis.” Science World, 28 July 2020, www.scienceworld.ca/

resource/naked-eggs-osmosis.

Weik, Matt. “IIFYM (If It Fits Your Macros).” Macro Diet Plan for Fast Weight Loss • IIFYM •

Calculate Your Macros, IIFYM, 21 July 2020, www.iifym.com/iifym-calculator/pre-

workout-supplements-6-side-effects-and-how-to-avoid-them. Accessed 18 Apr. 2021.

Zavvos, Athanasios, et al. “The Effect of Caffeine Intake on Body Fluids Replacement After

Exercise-Induced Dehydration.” Nutrition Today, vol. 55, no. 6, 2020, p. 288. Crossref,

doi:10.1097/nt.0000000000000445. Accessed 14 Apr. 2021.

15

Zhang, Yang, et al. “Caffeine and Diuresis during Rest and Exercise: A Meta-Analysis.” Journal

of Science and Medicine in Sport, vol. 18, no. 5, 2015, pp. 570–573. Crossref,

doi:10.1016/j.jsams.2014.07.017. Accessed 14 Apr. 2021

16

Appendix:

Table 1: Raw Data for the Study

Table 1: This table shows the raw data collected in the lab for each trial as well as the percent

change in weight in the naked eggs.

Sample Number

Caffeine Content (mg)

Caffeine Concentration (mg/mL)

Initial Weight of the Naked Egg (mg)

Final Weight of the Naked egg (mg)

Percent change in weight

1 0 0 87.3 99.2 13.6

2 0 0 92.2 105.2 14.1

3 0 0 89.4 97.4 8.9

4 200 0.4 88.7 98.9 11.5

5 200 0.4 82.2 93.6 13.9

6 200 0.4 90.9 108.9 19.8

7 400 0.8 85.8 100.0 16.6

8 400 0.8 87.0 99.4 14.3

9 400 0.8 92.3 105.0 13.8

10 600 1.2 83.9 98.8 17.8

11 600 1.2 87.3 102.1 16.9

12 600 1.2 84.4 95.0 12.6

17

Figure 4: This figure shows the QQ plot of the raw percent change data from table 1. The data

did not undergo any transformation so the percent change was plotted just as it was shown in

table 1. The data fits the line of best fit and is therefore normally distributed.

18

Figure 5: This figure shows the p-value obtained from the one-way ANOVA test using graphpad

prism 9. This p-value was greater than the significance level of 0.05 in the study so the data did

not yield significant results and the null hypothesis could not be rejected and the alternate

hypothesis could not be supported.

19

Figure 6: Shows the p-values obtained from the Shapiro-Wilk test using graphpad prism 9. The

p-values are greater than the significance level of 0.05 therefore, the data obtained followed a

normal distribution.

Equation 2: The percent change in weight experienced by the naked eggs was calculated using

the above formula.

Equation 3: This equation shows how the caffeine concentrations used in this study were

calculated.

Percent Change in Weight  = (Final Weight (g) − Init ial Weight (g))/Init ial Weight x 100%

Concentration (mg /mL) = Amount of Caf feine in Solution(mg) ∕ Volume of the Solution(mL)